TCEP relies on a completely different chemistry to achieve the same goal as BME or DTT. DTT/BME use S(-)>SS with redox potentials of -0.26 to -0.33 V (at pH 7) whereasTCEP uses P(3+)->P(5+) oxidation with redox potential that's a lot higher (I don't know of a reference with a stated redox potential for this system) because TCEP readily reduces oxidized forms of both DTT and BME in solution. TCEP also does not readly break S-Hg bonds unlike DTT or BME.
TCEP does not readily react with oxygen in solution (both DTT and BME react rapidly) and has long shelf life as buffered 1M solution at pH 5.6-6.3. TCEP has two non-trivial disadvantages that are for the most part not relevant for the purposes of crystallography: one is that it does not work very well in high Phosphate concentration, and the other is that it hinders the reaction of thiols with haloacetamides and suchlike (because it itself can react with haloacetamides). If you're labeling with haloacetamides you may want to use tri- tertbutul phosphine instead (it's much less soluble than TCEP, but 1 mM solution in water can be made). TCEP does not permeate biological membranes and therefore has been used to reduce thiols outside the cell while keeping intracellular ones intact. Due to its size and charge, it also is quite selective which protein disulphides are readily reduced - ones on or near protein surface are reduced quickly whereas buried or shielded ones are often not reduced at all w/o the use of a chaotrope. That's rather useful to us as it often allows us to reduce the unwanted inter-molecular disulphides (bad: aggregates) while at the same time preserve the valuable intra-molecular ones (good: structure) Artem On Sat, Apr 16, 2011 at 11:46 PM, Nian Huang <[email protected]> wrote: > Dear Horacio, > How does TECEP compare to BME or DTT? People claim it is better, but I > want some crystallographers' opinion? > > Nian > > On Sat, Apr 16, 2011 at 4:24 PM, Horacio Botti <[email protected]>wrote: > >> Dear Mike >> >> BME readily autooxidizes (need for metal traces and dissolved O2). Is >> yours a metalloprotein? Is your buffer contaminated with metals? Those >> situations would make the case a bit different. If not, unless your BME >> stock is already oxidized, blocking of the accesible thiols with BME should >> take some time. If you treat your protein for 40 min with fresh BME you >> should not observe thiol blocking. If you let the preparation to stay for >> several days, even at 4-6 °C you may observe the blocking that you may be >> observing. >> >> If you want to prevent Cys blocking you can also change to DTT (it is a >> dithiol, does not readily form mixed disulfides) and use it with caution >> (for thiol reduction it is advisable to use stoichiometric DTT (with respect >> to the number of Cys you need to reduce) and 10 fold excess of BME, look for >> their redox potentials). Take care of not "over-reducing" your protein if >> internal disulfide bonds are expected. Once reduced I suggest you to remove >> any reducing agent and store the protein at -80 °C. >> >> External Cys can be easily oxidized, they are highly expossed to metals >> and oxidants (H2O2, BME disulfides, etc). Diffusion is for sure much faster >> than SS bond formation, although some cys react at almost >> diffusion-controlled rates with oxidants (is yours a thiol'dependen t >> peroxidase?) You can take a look at the following reference (advertising): >> >> 2011. Factors Affecting Protein Thiol Reactivity and Specificity in >> Peroxide Reduction. Chem Res Toxicol. >> >> Metals can contaminate bad quality materials (water, salts, buffers, etc), >> take care of that too. If you need to control the redox state of your >> protein you should use DTNB (Ellman´s reagent), or DTDPy, to measure >> accesible reduced thiol groups. >> >> Good luck! >> >> Horacio >> >> >> >> >> Quoting Kendall Nettles <[email protected]>: >> >> We see BME adducts in all of our estrogen receptor structures, though we >>> don't always put them in the models. Sometimes we only see one or two atoms >>> of the adduct, and in others it is completely ordered. We only see it on >>> the solvent accessible cysteines. We do it on purpose. We used to treat the >>> protein with iodoacetic acid to generate uniform modification of the >>> cysteines, but then we realized we could get then same homogeneity with >>> 20-50mM BME. >>> >>> Kendall Nettles >>> >>> On Apr 15, 2011, at 4:09 PM, "Michael Thompson" <[email protected]> >>> wrote: >>> >>> Hi All, >>>> >>>> I was wondering if anyone knew whether or not it is possible for >>>> reducing agents with thiol groups, such as DTT or beta-mercaptoethanol >>>> (BME), to form covalent S-S bonds with Cys residues, particularly >>>> solvent-exposed Cys? I have some puzzling biochemical results, and in the >>>> absence of a structure (thus far), I was wondering if this might be >>>> something to try to control for. I have never heard of this happening (or >>>> seen a structure where there was density for this type of adduct), but I >>>> can't really think of a good reason for why this wouldn't happen. >>>> Especially for something like BME, where the molecule is very much like >>>> the >>>> Cys sidechain and seems to me like it should have similar reactivity. The >>>> only thing I can think of is if there is a kinetic effect taking place. >>>> Perhaps the rate of diffusion of these small molecules is much faster >>>> that >>>> the formation of the S-S bond? >>>> >>>> Does anyone know whether or not this is possible, and why it does or >>>> does not happen? >>>> >>>> Thanks, >>>> >>>> Mike >>>> >>>> >>>> >>>> >>>> -- >>>> Michael C. Thompson >>>> >>>> Graduate Student >>>> >>>> Biochemistry & Molecular Biology Division >>>> >>>> Department of Chemistry & Biochemistry >>>> >>>> University of California, Los Angeles >>>> >>>> [email protected] >>>> >>> >>> >
